PROJECT SUMMARY The long-term goal of our research program is to understand mechanisms of human metapneumovirus (HMPV) immunity and pathogenesis and facilitate vaccine development. HMPV is a leading cause of severe lower respiratory infection (LRI) in children and adults worldwide. There is no licensed vaccine against HMPV. Recurrent infections with HMPV and other respiratory viruses such as respiratory syncytial virus (RSV) occur throughout life. Consequently, HMPV and RSV cause severe illness in older adults and persons with underlying conditions such as asthma, immune compromise, and chronic cardiopulmonary disease. Limited induction of CD8+ T cell (TCD8) memory by respiratory viruses may be a contributing factor to reinfection and presents an obstacle to the development of effective vaccines. RSV, influenza, and parainfluenza viruses have been shown to induce TCD8 with impaired cytolytic function and IFN? secretion, but the mechanism of this inhibition was unknown. Similar antigen unresponsiveness termed TCD8 exhaustion is associated with chronic viral infections and cancer. A key mediator of exhausted TCD8 is programmed cell death-1 (PD-1), a negative regulator of T cell activation implicated in maintaining peripheral tolerance and preventing autoimmunity. PD-1 and other inhibitory receptors including LAG-3 are therapeutic targets in chronic infections and cancer. A recognized adverse effect in these patients is respiratory inflammation, likely due to over-activated TCD8. We discovered that HMPV, influenza, and other respiratory viruses induce TCD8 impairment mediated initially by the PD-1 pathway. However, our preliminary data suggest that other inhibitory receptors, including LAG-3, are involved in maintaining lung TCD8 impairment in later stages of exhaustion. We hypothesize that respiratory viruses activate an orchestrated network of inhibitory pathways, which limit the acute TCD8 response and immunopathology, but also limit the induction of effective memory TCD8. These inhibitory pathways are likely natural immunoregulatory responses to limit lung inflammation, but which also limit effective host response and memory. Elucidating these mechanisms would increase understanding of poor immune memory against respiratory viruses and help guide vaccine development. In Specific Aim 1, we will test the hypothesis that PD-L1 has distinct functions on different cell types in the lung. In Aim 2, we will define the contribution of other inhibitory receptor pathways to late TCD8 impairment. In Specific Aim 3, we will test the hypothesis that lung TCD8 impairment programming can be bypassed using non-replicating virus-like particle (VLP) vaccines with local inhibitory receptor blockade. This work will elucidate mechanisms of TCD8 impairment and define the contribution of TCD8 immunoregulatory pathways in the memory response to vaccination and viral respiratory infections. The findings will guide HMPV vaccine development and identify biomarkers of effective TCD8 responses to vaccines. The results of these experiments will be applicable to other respiratory pathogens as well as relevant to respiratory complications of inhibitory receptor blockade therapy.